EP2294225B1 - Verfahren zur direkten amplifikation aus rohen nukeinsäureproben - Google Patents

Verfahren zur direkten amplifikation aus rohen nukeinsäureproben Download PDF

Info

Publication number
EP2294225B1
EP2294225B1 EP09774388.4A EP09774388A EP2294225B1 EP 2294225 B1 EP2294225 B1 EP 2294225B1 EP 09774388 A EP09774388 A EP 09774388A EP 2294225 B1 EP2294225 B1 EP 2294225B1
Authority
EP
European Patent Office
Prior art keywords
paper
pcr
direct buffer
blood
nucleic acid
Prior art date
Legal status (The legal status is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the status listed.)
Active
Application number
EP09774388.4A
Other languages
English (en)
French (fr)
Other versions
EP2294225A2 (de
EP2294225A4 (de
EP2294225B8 (de
Inventor
Chien-Wei Chang
Dennis Y. Wang
Lori K. Hennessy
Current Assignee (The listed assignees may be inaccurate. Google has not performed a legal analysis and makes no representation or warranty as to the accuracy of the list.)
Life Technologies Corp
Original Assignee
Life Technologies Corp
Priority date (The priority date is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the date listed.)
Filing date
Publication date
Application filed by Life Technologies Corp filed Critical Life Technologies Corp
Priority to HRP20150280AT priority Critical patent/HRP20150280T1/hr
Priority to SI200931149T priority patent/SI2294225T1/sl
Priority to EP14197738.9A priority patent/EP2905344B1/de
Publication of EP2294225A2 publication Critical patent/EP2294225A2/de
Publication of EP2294225A4 publication Critical patent/EP2294225A4/de
Publication of EP2294225B1 publication Critical patent/EP2294225B1/de
Application granted granted Critical
Publication of EP2294225B8 publication Critical patent/EP2294225B8/de
Active legal-status Critical Current
Anticipated expiration legal-status Critical

Links

Images

Classifications

    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12Q—MEASURING OR TESTING PROCESSES INVOLVING ENZYMES, NUCLEIC ACIDS OR MICROORGANISMS; COMPOSITIONS OR TEST PAPERS THEREFOR; PROCESSES OF PREPARING SUCH COMPOSITIONS; CONDITION-RESPONSIVE CONTROL IN MICROBIOLOGICAL OR ENZYMOLOGICAL PROCESSES
    • C12Q1/00—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions
    • C12Q1/68—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions involving nucleic acids
    • C12Q1/6876—Nucleic acid products used in the analysis of nucleic acids, e.g. primers or probes
    • C12Q1/6888—Nucleic acid products used in the analysis of nucleic acids, e.g. primers or probes for detection or identification of organisms
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12Q—MEASURING OR TESTING PROCESSES INVOLVING ENZYMES, NUCLEIC ACIDS OR MICROORGANISMS; COMPOSITIONS OR TEST PAPERS THEREFOR; PROCESSES OF PREPARING SUCH COMPOSITIONS; CONDITION-RESPONSIVE CONTROL IN MICROBIOLOGICAL OR ENZYMOLOGICAL PROCESSES
    • C12Q1/00—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions
    • C12Q1/68—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions involving nucleic acids
    • C12Q1/6806—Preparing nucleic acids for analysis, e.g. for polymerase chain reaction [PCR] assay
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12Q—MEASURING OR TESTING PROCESSES INVOLVING ENZYMES, NUCLEIC ACIDS OR MICROORGANISMS; COMPOSITIONS OR TEST PAPERS THEREFOR; PROCESSES OF PREPARING SUCH COMPOSITIONS; CONDITION-RESPONSIVE CONTROL IN MICROBIOLOGICAL OR ENZYMOLOGICAL PROCESSES
    • C12Q1/00—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions
    • C12Q1/68—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions involving nucleic acids
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12Q—MEASURING OR TESTING PROCESSES INVOLVING ENZYMES, NUCLEIC ACIDS OR MICROORGANISMS; COMPOSITIONS OR TEST PAPERS THEREFOR; PROCESSES OF PREPARING SUCH COMPOSITIONS; CONDITION-RESPONSIVE CONTROL IN MICROBIOLOGICAL OR ENZYMOLOGICAL PROCESSES
    • C12Q1/00—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions
    • C12Q1/68—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions involving nucleic acids
    • C12Q1/6844—Nucleic acid amplification reactions
    • C12Q1/686—Polymerase chain reaction [PCR]

Definitions

  • the present teachings generally relate to methods of directly amplifying nucleic acids from crude samples.
  • RNA isolation and purification methods and kits are commercially available.
  • WO 00/50564 A2 relates to a composition and to a method for extracting DNA.
  • WO 2008/002740 A2 provides methods and compositions for extracting RNA from cells.
  • WO 2006/090987 A1 describes methods for performing a direct enzymatic reaction involving a nucleic acid molecule, and uses a zwitterionic buffer and/or a non-reducing carbohydrate to prevent the biological specimen from inhibiting the enzymatic reaction.
  • EP 1 050 587 B1 describes compositions and methods for the generation of nucleic acids from a ribonucleic acid template and further nucleic acid replication. However, their use adds time and expense to the preparation of samples for subsequent analysis.
  • the invention can be defined by the appending claims.
  • the invention provides a method according to claims 1-5, 7 and 9.
  • the invention provides a reaction mixture according to claims 6 and 8.
  • the present teachings provide a method of performing a polymerase chain reaction (PCR) comprising; providing a crude sample comprising: deoxyribonucleic acid; optionally incubating the crude sample with NaOH at 5 mM to 25mM; mixing the crude sample with a direct buffer to form a nucleic acid containing solution; and performing a PCR on the deoxyribonucleic acid, wherein the direct buffer comprises at least 3%-8% glycerol, 0.2%-0.9% non-ionic surfactants, and 1000-3000 ug/ml BSA.
  • Suitable non-ionic surfactants include, but not limited to, polysorbate (Tween), Tween 20, Triton-X 100 and the like.
  • the present teachings provide methods of performing a polymerase chain reaction (PCR) comprising; providing a crude sample comprising: deoxyribonucleic acid; optionally incubating the crude sample with NaOH at 5 mM to 25 mM; mixing the crude sample with a direct buffer to form a nucleic acid containing solution; and performing PCR on the solution, wherein the direct buffer comprises at least 5 PCR primer pairs, 10-50 mM Tris-HCl (pH 8.3), 30-80 mM KCI, 1.4-2.4 mM MgCl 2 , 0.01%-0.04% Sodium azide, 3%-8% glycerol, 100-350 uM of each dNTPs, 0.2%-0.9% polysorbate, 1000-3000 ug/ml BSA, and 0.10-0.35 U/ul of DNA polymerase.
  • PCR polymerase chain reaction
  • the present teachings provide methods of determining the identity of a human comprising; providing a crude sample comprising deoxyribonucleic acid from the human; optionally incubating the crude sample with NaOH at 5 mM to 25 mM; mixing the crude sample with the direct buffer forming a nucleic acid containing solution; wherein the direct buffer comprises a plurality of primer pairs; wherein each primer pair flanks a genomic locus containing a short tandem repeat (STR); performing PCR on the solution to form a plurality of PCR amplicons; wherein each PCR amplicon has an ascertainable size; and identifying the human by reference to size of the PCR amplicons; wherein the direct buffer further comprises 10-50 mM Tris-HCl (pH 8.3), 30-80 mM KCI, 1.4-2.4 mM MgCl 2 , 0.01%-0.04% Sodium azide, 3%-8% glycerol, 100-350 uM of each dNTPs, 0.2%-0.9
  • the embodiments include methods of preparing nucleic acids for a downstream enzymatic manipulation comprising; providing a crude sample comprising: deoxyribonucleic acid; optionally incubating the crude sample with NaOH at 5 mM to 25 mM; mixing the crude sample with a direct buffer to form a nucleic acid containing solution; and performing a downstream enzymatic manipulation on the solution, wherein the direct buffer comprises 10-50 mM Tris-HCl (pH 8.3), 30-80 mM KCl, 1.4-2.4 mM MgCl 2 , 0.01%-0.04% Sodium azide, 3%-8% glycerol, 100-350 uM of each dNTPs, 0.2%-0.9% polysorbate, 1000-3000 ug/ml BSA, and 0.10-0.35U/ul of DNA polymerase.
  • the downstream enzymatic manipulation is PCR.
  • kits for genetic analysis comprising; a plurality of primer pairs, wherein each primer pair flanks a genomic locus containing a short tandem repeat (STR); and a direct buffer, wherein the direct buffer comprises 10-50 mM Tris-HCl (pH 8.3), 30-80 mM KCI, 1.4-2.4 mM MgCl 2 , 0.01%-0.04% sodium azide, 3%-8% glycerol, 100-350 uM of each dNTPs, 0.2%-0.9% polysorbate, 1000-3000 ug/ml BSA, and 0.10 -0.35U/ul of DNA polymerase.
  • the kits as set forth above can optionally include NaOH or other strong alkaline compounds, or other agents for cell lysis.
  • kits comprising; a direct buffer and a plurality of primer pairs, wherein each primer pair flanks a genomic locus containing a short tandem repeat (STR), and wherein the direct buffer comprises 10-50 mM Tris-HCl (pH 8.3), 30-80 mM KCI, 1.4-2.4 mM MgCl 2 , 0.01%-0.04% Sodium azide, 3%-8% glycerol, 100-350 uM of each dNTPs, 0.2%-0.9% polysorbate, 1200-3000 ug/ml BSA, and 0.10 - 0.35 U/ul of DNA polymerase.
  • the kits as set forth above can optionally include NaOH or other strong alkaline compounds, or other agents for cell lysis.
  • the term "crude sample” refers to a specimen of biological origin suspected of containing nucleic acids, which has not undergone procedures for the isolation or purification of those nucleic acids.
  • a sample of blood is a crude sample.
  • the cells in crude sample can optionally be lysed.
  • a sample of blood spotted on paper containing a lysis reagent, such as FTA paper (commercially available from Whatman) is a crude sample.
  • a buccal swab of cheek cells is another example of a crude sample.
  • Crude samples include, but are not limited to, blood, diluted blood, blood on paper, buccal swabs, and buccal swabs on a substrate for samples storage, such as FTA paper.
  • FTA paper commercially available from Whatman
  • direct buffer refers to a buffer into which a crude sample can be placed.
  • the direct buffer contains primers and enzyme for performing a downstream enzymatic manipulation, such as a polymerase chain reaction (PCR).
  • PCR polymerase chain reaction
  • the direct buffer allows for the liberation of the nucleic acids and for their amplification directly in the direct buffer, without the need for any nucleic acid isolation or purification. Illustrative cycling times and temperatures for PCR can be found in Sambrook et al., Molecular Cloning, 3rd Edition 1993 .
  • the direct buffer of the present teachings focus on the use of the direct buffer for PCR, it will be appreciated that one of skill in the art can easily employ the direct buffer of the present teachings as a front-end procedure for other types of downstream enzymatic manipulations, for example reverse transcription using a reverse transcriptase, or an oligonucleotide ligation assay using a ligase.
  • downstream when used in reference to methods and manipulations performed on a target nucleic acids refers to methods and manipulations performed on a target nucleic acid sample subsequent to a method for liberating a nucleic acid from a biological sample, including, but not limited to, lysis of a cell to release the nucleic acid from the cell.
  • downstream enzymatic manipulation refers to procedures performed on a nucleic acid sample, including, but not limited to PCR using a polymerase, reverse transcription using a reverse transcriptase, or an oligonucleotide ligation assay using a ligase.
  • strong alkaline compound as used herein includes, but is not limited to, NaOH. Strong alkaline compounds can be used to lyse cells prior to the addition of some embodiments of direct buffer.
  • genomic DNA refers to the chromosomal DNA sequence of a gene or segment of a gene, including the DNA sequences of non-coding as well as coding regions. Genomic DNA also refers to DNA isolated directly from cells or chromosomes or the cloned copies of all or part of such DNA.
  • allele refers to a genetic variation associated with a gene or a segment of DNA, i.e., one of two or more alternate forms of a DNA sequence occupying the same locus.
  • locus refers to a specific position on a chromosome or a nucleic acid molecule. Alleles of a locus are located at identical sites on homologous chromosomes.
  • STR locus refers to any region of the human genome which contains short, repetitive sequence elements of 3 to 7 base pairs in length.
  • genomic locus containing a short tandem repeat refers to an STR locus in which the number of repetitive sequence elements (and net length of sequence) in a particular region of genomic DNA varies from allele to allele, and from individual to individual.
  • amplification primer and “primer” refers to an oligonucleotide, which is capable of site-specifically annealing to an RNA or DNA region adjacent a target sequence, and serving as an initiation primer for DNA synthesis under suitable conditions in which synthesis of a primer extension product is induced, e.g., in the presence of nucleotides and a polymerization-inducing agent such as a DNA-dependent DNA polymerase and at suitable temperature, pH, metal concentration, and salt concentration.
  • a polymerization-inducing agent such as a DNA-dependent DNA polymerase and at suitable temperature, pH, metal concentration, and salt concentration.
  • a PCR reaction employs a pair of amplification primers also known as “primer pairs” including an "upstream” or “forward” primer and a “downstream” or “reverse” primer, which delimit a region of the RNA or DNA to be amplified.
  • primer pairs also known as “primer pairs” including an "upstream” or “forward” primer and a “downstream” or “reverse” primer, which delimit a region of the RNA or DNA to be amplified.
  • amplifying refers to a process whereby a portion of a nucleic acid is replicated using, for example, any of a broad range of primer extension reactions.
  • primer extension reactions include, but are not limited to, PCR.
  • amplifying refers to a single replication or to an arithmetic, logarithmic, or exponential amplification.
  • amplicon and "PCR amplicon” as used herein refers to a broad range of techniques for increasing polynucleotide sequences, either linearly or exponentially and can be the product of an amplification reaction.
  • An amplicon can be double-stranded or single-stranded, and can include the separated component strands obtained by denaturing a double-stranded amplification product.
  • the amplicon of one amplification cycle can serve as a template in a subsequent amplification cycle.
  • Exemplary amplification techniques include, but are not limited to, PCR or any other method employing a primer extension step.
  • amplification examples include, but are not limited to, ligase detection reaction (LDR) and ligase chain reaction (LCR).
  • LDR ligase detection reaction
  • LCR ligase chain reaction
  • Amplification methods can comprise thermal-cycling or can be performed isothermally.
  • the term "amplification product" includes products from any number of cycles of amplification reactions.
  • nucleic acid sequence can refer to the nucleic acid material itself and is not restricted to the sequence information (i.e. the succession of letters chosen among the five base letters A, C, G, T, or U) that biochemically characterizes a specific nucleic acid, for example, a DNA or RNA molecule.
  • polynucleotide refers to a linear polymer of natural or modified monomers or linkages, including deoxyribonucleic acid, deoxyribonucleosides, ribonucleosides, polyamide nucleic acids, and the like, joined by inter-nucleosidic linkages and have the capability of specifically binding to a target polynucleotide by way of a regular pattern of monomer-to-monomer interactions, such as Watson-Crick type of base pairing, and capable of being ligated to another oligonucleotide in a template-driven reaction.
  • oligonucleotides ranging in size from a few monomeric units, e.g. 3-4, to several hundreds of monomeric units.
  • a polynucleotide such as an oligonucleotide is represented by a sequence of letters, such as "ATGCCTG,” it will be understood that the nucleotides are in 5' ⁇ 3' order from left to right and that "A” denotes deoxyadenosine, "C” denotes deoxycytidine, “G” denotes deoxyguanosine, and "T” denotes deoxythymidine, unless otherwise noted.
  • A, C, G, and T can be used to refer to the bases themselves, to nucleosides, or to nucleotides comprising the bases, as is standard in the art.
  • the inter-nucleoside linkage is typically a phosphodiester bond, and the subunits are referred to as "nucleotides.”
  • sequence determination includes determination of partial as well as full sequence information. That is, the term includes sequence comparisons, fingerprinting, and like levels of information about a target polynucleotide, as well as the express identification and ordering of each nucleoside of the target polynucleotide within a region of interest.
  • sequence determination comprises identifying a single nucleotide, while in other embodiments more than one nucleotide is identified. Identification of nucleosides, nucleotides, and/or bases are considered equivalent herein. It is noted that performing sequence determination on a polynucleotide typically yields equivalent information regarding the sequence of a perfectly complementary polynucleotide and thus is equivalent to sequence determination performed directly on a perfectly complementary polynucleotide.
  • oligonucleotide probes includes sets of two or more oligonucleotide probes where there may be a single "common” oligonucleotide probe that is usually specific for a non-variable region of a target polynucleotide and one or more "wild-type” and/or “mutant” oligonucleotide probes that are usually specific for a region of a target polynucleotide that contains allelic or mutational variants in sequence.
  • Nucleic acid polymerase or “polymerase” refers to any polypeptide that catalyzes the synthesis of a polynucleotide using an existing polynucleotide as a template.
  • DNA polymerase refers to a nucleic acid polymerase that catalyzes the synthesis of DNA using an existing polynucleotide as a template.
  • the present teachings include direct buffers comprising 3%-8% glycerol, 0.2%-0.9% non-ionic surfactants, and 1200-3000 ug/ml BSA.
  • the present teachings include a direct buffers comprising 10-50 mM Tris-HCl (pH 8.3), 30-80 mM KCI, 1.4-2.4 mM MgCl 2 , 0.01%-0.04% Sodium azide, 3%-8% glycerol, 100-350 uM of each dNTPs, 0.2%-0.9% non-ionic surfactants, 1200-3000 ug/ml BSA, and 0.10l-0.35U/ul of DNA polymerase.
  • the non-ionic surfactants can be polysorbate (Tween, Tween 20), Triton-X 100 and the like.
  • DNA polymerase is commercially available from Applied Biosystems. Suitable DNA polymerases include Pol A, Pol B, Pol C, Pol D, Pol X, and Pol Y DNA polymerases and a Type I, Type II and Type III DNA polymerase. Generally, thermostable DNA polymerases such as Taq, Pfu, VentTMDeepVentTM, 9o NorthTM, are used in PCR. The DNA polymerases may be modified so as to be inactive prior to subsequent treatment, e.g. heat; for example see US patent 5,773,258 (chemically modified DNA polymerases), US patent 5,338,671 antibody bound DNA polymerases. DNA polymerases are known to those skilled in the art. DNA polymerases include DNA-dependent polymerases, which use DNA as a template, or RNA-dependent polymerases, such as reverse transcriptase, which use RNA as a template.
  • DNA-dependent DNA polymerases fall into one of six families (A, B, C, D, X, and Y), with most falling into one of three families (A, B, and C). See, e.g., Ito et al. (1991) Nucleic Acids Res. 19:4045-4057 ; Braithwaite et al. (1993) Nucleic Acids Res. 21:787-802 ; Filee et al. (2002) J. Mol. Evol. 54:763-773 ; and Alba (2001) Genome Biol. 2:3002.1-3002.4 .
  • DNA polymerases may be single-chain polypeptides (e.g., certain family A and B polymerases) or multi-subunit enzymes (e.g., certain family C polymerases) with one of the subunits having polymerase activity.
  • a fusion protein may comprise a DNA polymerase selected from a family A, B, C, D, X, or Y polymerase.
  • Polymer A Family A polymerases
  • Replicative members from this family include T7 DNA polymerase and the eukaryotic mitochondrial DNA Polymerase ⁇ .
  • repair polymerases are E. coli DNA Pol I, Thermus aquaticus Pol I (Taq DNA polymerase), and Bacillus stearothermophilus Pol I. Excision repair and processing of Okazaki fragments generated during lagging strand synthesis are performed by the repair polymerases Because most thermostable Pol A enzymes do not possess the 3' to 5' exonuclease activity, they are incapable of proofreading the newly synthesized nucleic acid strand and consequently have high error rates.
  • Poly B Family B polymerases
  • Pol B polymerases are substantially replicative polymerases including the major eukaryotic DNA polymerases ⁇ , ⁇ , ⁇ , and also DNA polymerase ⁇ .
  • Pol B polymerases also include DNA polymerases encoded by some bacteria and bacteriophages, of which the best characterized are from T4, Phi29 and RB69 bacteriophages.
  • Pol B enzymes are involved in both leading and lagging strand synthesis and are noteworthy for their remarkable accuracy during replication as many have strong 3'-5' exonuclease activity the exceptions being DNA polymerase ⁇ and ⁇ which lack proofreading activity.
  • BSA is commercially available from a variety of sources, for example catalog number 10711454001 from Roche.
  • FTA paper is commercially available from Whatman. In some embodiments, FTA paper is used herein with bloodstains. For example, Bloodstain Card from Whatman (Cat# WB 10 0014).
  • the FTA paper containing blood or buccal cells is cut out to remove a small region containing biological material, e.g., a circular punch of 0.5mm to 1.2mm in diameter, or1.0-1.5mm in diameter, or 1.5-2.0mm in diameter.
  • the paper is placed directly into the PCR mix comprising the direct buffer for PCR reaction. In some embodiments, a wash of the FTA paper is not required before placing the FTA paper in the direct buffer.
  • the cells when the cells are not initially lysed, e.g., when non-FTA paper is used, a NaOH solution at 5-25 mM is incubated with non-FTA before mixing with the direct buffer and performing a PCR reaction.
  • the cells may be lysed by exposure to reagents other than NaOH.
  • the direct buffer further comprises a plurality of PCR primer pairs.
  • the direct buffer comprises 5 primer pairs.
  • the direct buffer comprises 10 primer pairs.
  • the direct buffer comprises greater than 10 primer pairs.
  • the direct buffer does not comprise PCR primer pairs, but rather the PCR primer pairs are added at a separate time.
  • Some embodiments of the subject methods eliminate the need for a sample purification procedure prior to performing PCR.
  • the same protocol can be applied to both blood and buccal cell samples on FTA cards.
  • the physical sample handling is significantly reduced, therefore this can prevent possible cross-contamination between samples as well as eliminate the possibility of human errors.
  • the amount of time required from FTA card to STR profiles can be reduced by at least 11/2 hours.
  • the method is compatible with the current automation instruments available in the forensic market.
  • the costs associated with the purification of DNA from FTA can be eliminated.
  • the success rate of getting a full STR profile is comparable when compared to the traditional standard sample preparation method.
  • a sample of genomic DNA used directly with the Applied Biosystems Identi f iler HID v.1 Kit provides a full STR profile.
  • the STR profile is either partial or undetectable when blood from genomic DNA is taken from blood spotted on an FTA disc punch (1.2 mm punch) without washing the disc to remove materials which interfere with subsequent sample testing, e.g., STR profiling via direct PCR, see Figures 2A-2D .
  • FTA paper Whatman
  • a 0.5 mm disc punch from the FTA paper was made and placed into the direct buffer containing PCR primers from the commercially available Identifier® Human Identity Kit (Applied Biosystems). PCR was then performed.
  • TE buffer 10mM Tris-Cl and 0.1 mM EDTA at pH 8.0. 1 ul of diluted blood was used to set up a PCR in the direct buffer.
  • buccal swab samples were collected and placed in 500ul TE buffer. The resulting suspension was heated at 97°C for 5 minutes. 10 ul of the resulting suspension was used to set up a PCR in the direct buffer.
  • kits designed to expedite performing certain methods.
  • kits serve to expedite the performance of the methods of interest by assembling two or more components used in carrying out the methods.
  • kits may contain components in pre-measured unit amounts to minimize the need for measurements by end-users.
  • kits may include instructions for performing one or more methods of the present teachings.
  • the kit components are optimized to operate in conjunction with one another.
  • the present teachings provide a kit comprising; a plurality of primer pairs, wherein each primer pair flanks a genomic locus containing a short tandem repeat (STR); and a direct buffer, wherein the direct buffer comprises 10-50 mM Tris-HCl (pH 8.3), 30-80 mM KCI, 1.4-2.4 mM MgCl 2 , 0.01%-0.04% Sodium azide, 3%-8% glycerol, 100-350 uM of each dNTPs, 0.2%-0.9% non-ionic surfactants, 1200-3000 ug/ml BSA, and 0.10 -0.35 U/ul of AmpliTaq Gold DNA polymerase.
  • the direct buffer comprises 10-50 mM Tris-HCl (pH 8.3), 30-80 mM KCI, 1.4-2.4 mM MgCl 2 , 0.01%-0.04% Sodium azide, 3%-8% glycerol, 100-350 uM of each dNTPs, 0.
  • kits, methods and reaction mixtures provided by the present teachings can be used with procedures for multiplexed PCR of degraded samples, as found for example in WO05054515 to Dimsoski and Woo .
  • the direct buffer in the kit comprises 0.2%-0.9% polysorbate, 3%-8% glycerol, 1200-3000 ug/ml BSA.
  • the direct buffer in the kit further comprises 10-50 mM Tris-HCl (pH 8.3), 30-80 mM KCl, 1.4-2.4 mM MgCl 2 , 0.01%-0.04% Sodium azide, 3%-8% glycerol, 100-350 uM of each dNTPs, 0.2%-0.9% polysorbate, 1200-3000 ug/ml BSA, and 0.10 -0.35U/ul of AmpliTaq Gold DNA polymerase.
  • the direct buffer in the kit feature comprises NaOH at 5 mM-25 mM.
  • the NaOH is supplied in a vial separate from the direct buffer.

Landscapes

  • Chemical & Material Sciences (AREA)
  • Life Sciences & Earth Sciences (AREA)
  • Organic Chemistry (AREA)
  • Proteomics, Peptides & Aminoacids (AREA)
  • Zoology (AREA)
  • Wood Science & Technology (AREA)
  • Health & Medical Sciences (AREA)
  • Engineering & Computer Science (AREA)
  • Analytical Chemistry (AREA)
  • Molecular Biology (AREA)
  • Biochemistry (AREA)
  • Microbiology (AREA)
  • Biotechnology (AREA)
  • Biophysics (AREA)
  • Physics & Mathematics (AREA)
  • Genetics & Genomics (AREA)
  • Immunology (AREA)
  • Bioinformatics & Cheminformatics (AREA)
  • General Engineering & Computer Science (AREA)
  • General Health & Medical Sciences (AREA)
  • Chemical Kinetics & Catalysis (AREA)
  • Measuring Or Testing Involving Enzymes Or Micro-Organisms (AREA)
  • Preparation Of Compounds By Using Micro-Organisms (AREA)
  • Saccharide Compounds (AREA)

Claims (9)

  1. Ein Verfahren, umfassend:
    Bereitstellen von Blut auf Papier oder einem bukkalen Abstrich auf Papier, wobei das Papier ein Lysereagenz enthält;
    Platzieren des Bluts auf Papier oder des bukkalen Abstrichs auf Papier in einen direkten Puffer, um eine Nukleinsäure-enthaltende Lösung zu bilden; und
    Durchführen einer PCR mit der Nukleinsäure-enthaltenden Lösung, umfassend 0,2%-0,9% Polysorbat, 3%-8% Glycerol und 1000-3000 ug/ml BSA.
  2. Das Verfahren entsprechend Anspruch 1, wobei der direkte Puffer weiter 10-50 mM Tris-HCl (pH 8,3), 30-80 mM KCl, 1,4-2,4 mM MgCl2, 0,01%-0,04% Natriumazid, 100-350 uM jedes dNTPs und 0,10-0,35 U/ul der DNA Polymerase umfasst.
  3. Das Verfahren entsprechend Anspruch 1, wobei das Polysorbat Polysorbat 20 ist.
  4. Das Verfahren nach Anspruch 1, wobei der direkte Puffer weiter eine Vielzahl von Primerpaaren umfasst.
  5. Das Verfahren nach Anspruch 1, wobei humanes Blut oder ein humaner bukkaler Abstrich bereitgestellt werden, wobei jedes Primerpaar einen genomischen Locus flankiert, der einen Short-Tandem-Repeat enthält,
    wobei die PCR mit der Nukleinsäure-enthaltenden Lösung durchgeführt wird, um eine Vielzahl an PCR-Amplikons zu erhalten, wobei jedes PCR-Amplikon eine bestimmbare Größe hat; und das Verfahren weiter das Identifizieren des Menschen durch Bezug auf die Größe der PCR-Amplikons umfasst.
  6. Ein Reaktionsgemisch, umfassend einen direkten Puffer, Blut auf Papier oder einen bukkalen Abstrich auf Papier und eine Vielzahl von Primerpaaren, wobei das Papier ein Lysereagenz enthält und wobei der direkte Puffer 10-50 mM Tris-HCl (pH 8,3), 30-80 mM KCl, 1,4-2,4 mM MgCl2, 0,01%-0,04% Natriumazid, 3%-8% Glycerol, 100-350 uM jedes dNTPs, 0,2%-0,9% Polysorbat 20, 1200-3000 ug/ml BSA und 0,10-0,35 U/ul der DNA Polymerase umfasst.
  7. Das Verfahren nach Anspruch 1, wobei das Papier FTA-Papier ist.
  8. Das Gemisch nach Anspruch 6, wobei das Papier FTA-Papier ist.
  9. Das Verfahren nach Anspruch 1, wobei das Bereitstellen von Blut auf Papier oder einem bukkalen Abstrich auf Papier das Bereitstellen eines FTA-Papiers und das Platzieren von Blut oder das Sammeln von bukkalen Zellen auf dem Papier umfasst.
EP09774388.4A 2008-06-30 2009-06-30 Verfahren zur direkten amplifikation aus rohen nukeinsäureproben Active EP2294225B8 (de)

Priority Applications (3)

Application Number Priority Date Filing Date Title
HRP20150280AT HRP20150280T1 (hr) 2008-06-30 2009-06-30 Postupak za izravno pojačanje iz neprerađenih uzoraka nukleinske kiseline
SI200931149T SI2294225T1 (sl) 2008-06-30 2009-06-30 Postopek za neposredno amplifikacijo iz vzorcev surove nukleinske kisline
EP14197738.9A EP2905344B1 (de) 2008-06-30 2009-06-30 Verfahren zur direkten Amplifikation aus rohen Nukleinsäureproben

Applications Claiming Priority (2)

Application Number Priority Date Filing Date Title
US7711308P 2008-06-30 2008-06-30
PCT/US2009/049323 WO2010002938A2 (en) 2008-06-30 2009-06-30 Method for direct amplification from crude nucleic acid samples

Related Child Applications (2)

Application Number Title Priority Date Filing Date
EP14197738.9A Division EP2905344B1 (de) 2008-06-30 2009-06-30 Verfahren zur direkten Amplifikation aus rohen Nukleinsäureproben
EP14197738.9A Division-Into EP2905344B1 (de) 2008-06-30 2009-06-30 Verfahren zur direkten Amplifikation aus rohen Nukleinsäureproben

Publications (4)

Publication Number Publication Date
EP2294225A2 EP2294225A2 (de) 2011-03-16
EP2294225A4 EP2294225A4 (de) 2011-10-26
EP2294225B1 true EP2294225B1 (de) 2014-12-17
EP2294225B8 EP2294225B8 (de) 2015-02-18

Family

ID=41466583

Family Applications (2)

Application Number Title Priority Date Filing Date
EP09774388.4A Active EP2294225B8 (de) 2008-06-30 2009-06-30 Verfahren zur direkten amplifikation aus rohen nukeinsäureproben
EP14197738.9A Not-in-force EP2905344B1 (de) 2008-06-30 2009-06-30 Verfahren zur direkten Amplifikation aus rohen Nukleinsäureproben

Family Applications After (1)

Application Number Title Priority Date Filing Date
EP14197738.9A Not-in-force EP2905344B1 (de) 2008-06-30 2009-06-30 Verfahren zur direkten Amplifikation aus rohen Nukleinsäureproben

Country Status (10)

Country Link
US (4) US8173401B2 (de)
EP (2) EP2294225B8 (de)
JP (2) JP5770085B2 (de)
CN (1) CN102177250B (de)
BR (1) BRPI0915886A2 (de)
DK (1) DK2294225T3 (de)
ES (1) ES2532632T3 (de)
HR (1) HRP20150280T1 (de)
SI (1) SI2294225T1 (de)
WO (1) WO2010002938A2 (de)

Families Citing this family (32)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO2010002938A2 (en) 2008-06-30 2010-01-07 Life Technologies Corporation Method for direct amplification from crude nucleic acid samples
US9184099B2 (en) 2010-10-04 2015-11-10 The Board Of Trustees Of The Leland Stanford Junior University Biosensor devices, systems and methods therefor
NZ610129A (en) 2010-10-04 2014-08-29 Genapsys Inc Systems and methods for automated reusable parallel biological reactions
US9399217B2 (en) 2010-10-04 2016-07-26 Genapsys, Inc. Chamber free nanoreactor system
US9469871B2 (en) 2011-04-14 2016-10-18 Corporos Inc. Methods and apparatus for point-of-care nucleic acid amplification and detection
US8911941B2 (en) * 2011-04-14 2014-12-16 Kenneth J. Michlitsch Methods and apparatus for point-of-care nucleic acid amplification and detection
US9926596B2 (en) 2011-05-27 2018-03-27 Genapsys, Inc. Systems and methods for genetic and biological analysis
US8585973B2 (en) 2011-05-27 2013-11-19 The Board Of Trustees Of The Leland Stanford Junior University Nano-sensor array
US9567628B2 (en) 2011-06-08 2017-02-14 Life Technologies Corporation Polymerization of nucleic acids using proteins having low isoelectric points
FI3461807T3 (fi) 2011-06-08 2023-09-07 Life Technologies Corp Uudenlaisten detergenttien suunnittelu ja kehitys pcr-järjestelmissä käyttöä varten
US9518901B2 (en) * 2011-06-29 2016-12-13 Kabushiki Kaisha Dnaform Pretreatment method of biological sample, detection method of RNA, and pretreatment kit
WO2013006793A2 (en) 2011-07-06 2013-01-10 Quest Diagnostics Investments Incorporated Direct amplification and detection of viral and bacterial pathogens
CN106591103B (zh) 2011-12-01 2021-06-04 吉纳普赛斯股份有限公司 用于高效电子测序与检测的系统和方法
GB201201464D0 (en) 2012-01-27 2012-03-14 Secr Defence Analytical method
US9354159B2 (en) 2012-05-02 2016-05-31 Nanoscopia (Cayman), Inc. Opto-fluidic system with coated fluid channels
GB201219137D0 (en) * 2012-10-24 2012-12-05 Ge Healthcare Uk Ltd Direct nucleic acid amplification kit, reagent and method
WO2014072354A1 (en) * 2012-11-09 2014-05-15 Ge Healthcare Uk Limited Methods for one step nucleic acid amplification of non-eluted samples
WO2014145078A1 (en) 2013-03-15 2014-09-18 Verinata Health, Inc. Generating cell-free dna libraries directly from blood
EP2971141B1 (de) 2013-03-15 2018-11-28 Genapsys, Inc. Systeme für biologische analysen
EP2971109B1 (de) * 2013-03-15 2018-10-24 GE Healthcare UK Limited Verfahren zur einstufigen nukleinsäureamplifikation von nicht-eluierten proben
EP3539944A1 (de) 2013-10-25 2019-09-18 Life Technologies Corporation Neuartige verbindungen zur verwendung in pcr-systemen und anwendungen davon
WO2015089238A1 (en) 2013-12-11 2015-06-18 Genapsys, Inc. Systems and methods for biological analysis and computation
WO2015096063A1 (en) * 2013-12-25 2015-07-02 Coyote Bioscience Co., Ltd. Methods and systems for nucleic acid amplification
CN103789299B (zh) * 2014-01-09 2016-08-31 东北制药集团辽宁生物医药有限公司 一种进行全血基因组快速扩增的方法
EP3556864B1 (de) 2014-04-18 2020-12-09 Genapsys, Inc. Verfahren und systeme zur nukleinsäureamplifikation
JP2018502281A (ja) * 2014-11-20 2018-01-25 ジーイー・ヘルスケア・ユーケイ・リミテッド 固体支持体材料に固定された認知症及びアルツハイマー病関連バイオマーカーの検出
US12077810B2 (en) 2016-04-14 2024-09-03 T2 Biosystems, Inc. Methods and systems for amplification in complex samples
CN105925692B (zh) * 2016-05-16 2019-09-20 安陆市疾病预防控制中心 一种常温稳定的pcr预混液
US10544456B2 (en) 2016-07-20 2020-01-28 Genapsys, Inc. Systems and methods for nucleic acid sequencing
CA3076378A1 (en) 2017-09-21 2019-03-28 Genapsys, Inc. Systems and methods for nucleic acid sequencing
CN107937491A (zh) * 2017-11-30 2018-04-20 广州定康医疗科技有限公司 一种针对口腔拭子的基于pcr的snp基因分型方法
WO2025175212A1 (en) * 2024-02-15 2025-08-21 Life Technologies Corporation Methods for rapid extraction-free genotyping

Family Cites Families (29)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US5837453A (en) * 1992-05-13 1998-11-17 Geron Corporation Telomerase activity assays
EP0590327B1 (de) * 1992-09-11 2003-04-09 F. Hoffmann-La Roche Ag Nachweis von Nukleinsäuren im Blut
US5338671A (en) 1992-10-07 1994-08-16 Eastman Kodak Company DNA amplification with thermostable DNA polymerase and polymerase inhibiting antibody
JPH07110455B2 (ja) 1992-10-27 1995-11-29 住友電気工業株式会社 ウェハ固定装置
US7008771B1 (en) * 1994-09-30 2006-03-07 Promega Corporation Multiplex amplification of short tandem repeat loci
US6479235B1 (en) 1994-09-30 2002-11-12 Promega Corporation Multiplex amplification of short tandem repeat loci
US5843660A (en) * 1994-09-30 1998-12-01 Promega Corporation Multiplex amplification of short tandem repeat loci
CA2118048C (en) 1994-09-30 2003-04-08 James W. Schumm Multiplex amplification of short tandem repeat loci
FR2737223B1 (fr) * 1995-07-24 1997-09-12 Bio Merieux Procede d'amplification de sequences d'acide nucleique par deplacement, a l'aide d'amorces chimeres
US5773258A (en) 1995-08-25 1998-06-30 Roche Molecular Systems, Inc. Nucleic acid amplification using a reversibly inactivated thermostable enzyme
US5861251A (en) * 1996-10-15 1999-01-19 Bioneer Corporation Lyophilized reagent for polymerase chain reaction
US5962665A (en) * 1997-06-16 1999-10-05 Abbott Laboratories Nucleic acid primers and probes for detecting HIV-1 and HIV-2
US6238863B1 (en) * 1998-02-04 2001-05-29 Promega Corporation Materials and methods for indentifying and analyzing intermediate tandem repeat DNA markers
US6410725B1 (en) * 1999-02-26 2002-06-25 Myriad Genetics Inc. Method for extracting DNA from dried specimens
US6300069B1 (en) * 1999-05-03 2001-10-09 Qiagen Gmbh Generation and amplification of nucleic acids from ribonucleic acids
US6297005B1 (en) 1999-05-04 2001-10-02 Schering Corporation De novo priming activity of hepatitis C virus replicase
US20030049614A1 (en) * 1999-10-06 2003-03-13 Holly Hurlbut Hogrefe Compositions for dna amplification, synthesis, and mutagenesis
AU2002226053A1 (en) * 2000-12-12 2002-06-24 Invitrogen Corporation Compositions and methods for the release of nucleic acid molecules from solid matrices
US7198897B2 (en) * 2001-12-19 2007-04-03 Brandeis University Late-PCR
WO2005017173A1 (en) * 2003-07-29 2005-02-24 Sigma Aldrich Co. Methods and compositions for amplification of dna
US20050112591A1 (en) 2003-11-25 2005-05-26 Applera Corporation Novel method for isolating single stranded product
US7776530B2 (en) * 2004-06-29 2010-08-17 Wallac Oy Integrated nucleic acid analysis
US20060014190A1 (en) * 2004-06-30 2006-01-19 Hennessy Lori K Methods for analyzing short tandem repeats and single nucleotide polymorphisms
EP1874955A4 (de) * 2005-02-28 2009-05-06 Bioquest Inc Verfahren zur durchführung direkter enzymatischer reaktionen unter beteiligung von nukleinsäuremolekülen
US20080003575A1 (en) * 2006-06-28 2008-01-03 Sigma-Aldrich Co. Methods and composition for RNA extraction
US20080124768A1 (en) * 2006-10-31 2008-05-29 Sigma Aldrich Co. Methods and Compositions for Amplification of DNA
US20080138804A1 (en) * 2006-12-12 2008-06-12 Helicos Biosciences Corporation Buffer composition
WO2009108170A2 (en) * 2007-11-16 2009-09-03 Invitrogen Corporation Compositions and methods for determining immune status
WO2010002938A2 (en) 2008-06-30 2010-01-07 Life Technologies Corporation Method for direct amplification from crude nucleic acid samples

Non-Patent Citations (2)

* Cited by examiner, † Cited by third party
Title
ABU AL-SOUD WALEED ET AL: "Effects of amplification facilitators on diagnostic PCR in the presence of blood, feces, and meat", JOURNAL OF CLINICAL MICROBIOLOGY, vol. 38, no. 12, December 2001 (2001-12-01), pages 4463 - 4470, ISSN: 0095-1137 *
NAGAI M ET AL: "ADDITIVE EFFECTS OF BOVINE SERUM ALBUMIN, DITHIOTHREITOL, AND GLYCEROL ON PCR", BIOCHEMISTRY AND MOLECULAR BIOLOGY INTERNATIONAL, ACADEMIC PRESS, LONDON, GB, vol. 44, no. 1, 1 January 1998 (1998-01-01), pages 157 - 163, XP009023284, ISSN: 1039-9712 *

Also Published As

Publication number Publication date
US8460934B2 (en) 2013-06-11
HRP20150280T1 (hr) 2015-04-10
DK2294225T3 (en) 2015-03-30
US8173401B2 (en) 2012-05-08
US20140120541A1 (en) 2014-05-01
JP2011526788A (ja) 2011-10-20
WO2010002938A2 (en) 2010-01-07
US20130005609A1 (en) 2013-01-03
EP2294225A2 (de) 2011-03-16
ES2532632T3 (es) 2015-03-30
WO2010002938A3 (en) 2010-04-08
CN102177250B (zh) 2015-05-06
SI2294225T1 (sl) 2015-04-30
US20150354015A1 (en) 2015-12-10
CN102177250A (zh) 2011-09-07
BRPI0915886A2 (pt) 2015-11-03
JP5770085B2 (ja) 2015-08-26
JP2014166189A (ja) 2014-09-11
EP2294225A4 (de) 2011-10-26
EP2905344A1 (de) 2015-08-12
US9518302B2 (en) 2016-12-13
US9057110B2 (en) 2015-06-16
EP2905344B1 (de) 2016-11-09
EP2294225B8 (de) 2015-02-18
US20100015621A1 (en) 2010-01-21

Similar Documents

Publication Publication Date Title
EP2294225B1 (de) Verfahren zur direkten amplifikation aus rohen nukeinsäureproben
US20230099446A1 (en) Fast pcr for str genotyping
JP6970205B2 (ja) Ｄｎａおよびｒｎａの同時濃縮を含むプライマー伸長標的濃縮およびそれに対する向上
CN107541546B (zh) 用于标靶核酸富集的组合物、方法、系统和试剂盒
CN117778531A (zh) 分子库制备方法以及其组合物和用途
CN113490751A (zh) 核酸检测和引物设计方法
US8153401B2 (en) Method for direct amplification from crude nucleic acid samples
US10246702B1 (en) Compositions and methods for labeling target nucleic acid molecules
Dada et al. Polymerase chain reaction
WO2022144003A1 (zh) 一种用于高通量靶向测序的多重pcr文库构建方法
CN116964221A (zh) 阻止测序期间核酸模板穿过纳米孔的结构
JP2022545779A (ja) 配列決定用のｄｎａ及びｒｎａのシングルチューブ調製

Legal Events

Date Code Title Description
PUAI Public reference made under article 153(3) epc to a published international application that has entered the european phase

Free format text: ORIGINAL CODE: 0009012

17P Request for examination filed

Effective date: 20110105

AK Designated contracting states

Kind code of ref document: A2

Designated state(s): AT BE BG CH CY CZ DE DK EE ES FI FR GB GR HR HU IE IS IT LI LT LU LV MC MK MT NL NO PL PT RO SE SI SK TR

AX Request for extension of the european patent

Extension state: AL BA RS

DAX Request for extension of the european patent (deleted)
A4 Supplementary search report drawn up and despatched

Effective date: 20110922

RIC1 Information provided on ipc code assigned before grant

Ipc: C12Q 1/68 20060101AFI20110916BHEP

17Q First examination report despatched

Effective date: 20130729

GRAP Despatch of communication of intention to grant a patent

Free format text: ORIGINAL CODE: EPIDOSNIGR1

INTG Intention to grant announced

Effective date: 20140702

GRAS Grant fee paid

Free format text: ORIGINAL CODE: EPIDOSNIGR3

GRAA (expected) grant

Free format text: ORIGINAL CODE: 0009210

AK Designated contracting states

Kind code of ref document: B1

Designated state(s): AT BE BG CH CY CZ DE DK EE ES FI FR GB GR HR HU IE IS IT LI LT LU LV MC MK MT NL NO PL PT RO SE SI SK TR

REG Reference to a national code

Ref country code: GB

Ref legal event code: FG4D

RAP2 Party data changed (patent owner data changed or rights of a patent transferred)

Owner name: LIFE TECHNOLOGIES CORPORATION

REG Reference to a national code

Ref country code: CH

Ref legal event code: EP

REG Reference to a national code

Ref country code: IE

Ref legal event code: FG4D

REG Reference to a national code

Ref country code: AT

Ref legal event code: REF

Ref document number: 701978

Country of ref document: AT

Kind code of ref document: T

Effective date: 20150115

RIN2 Information on inventor provided after grant (corrected)

Inventor name: HENNESSY, LORI, K.

Inventor name: WANG, DENNIS, Y.

Inventor name: CHANG, CHIEN-WEI

REG Reference to a national code

Ref country code: DE

Ref legal event code: R096

Ref document number: 602009028415

Country of ref document: DE

Effective date: 20150205

REG Reference to a national code

Ref country code: HR

Ref legal event code: TUEP

Ref document number: P20150280

Country of ref document: HR

REG Reference to a national code

Ref country code: NL

Ref legal event code: T3

REG Reference to a national code

Ref country code: DK

Ref legal event code: T3

Effective date: 20150324

Ref country code: ES

Ref legal event code: FG2A

Ref document number: 2532632

Country of ref document: ES

Kind code of ref document: T3

Effective date: 20150330

REG Reference to a national code

Ref country code: SE

Ref legal event code: TRGR

Ref country code: CH

Ref legal event code: NV

Representative=s name: PATENTANWALT MATTHIAS W. SCHAEFER, CH

REG Reference to a national code

Ref country code: HR

Ref legal event code: T1PR

Ref document number: P20150280

Country of ref document: HR

PG25 Lapsed in a contracting state [announced via postgrant information from national office to epo]

Ref country code: LT

Free format text: LAPSE BECAUSE OF FAILURE TO SUBMIT A TRANSLATION OF THE DESCRIPTION OR TO PAY THE FEE WITHIN THE PRESCRIBED TIME-LIMIT

Effective date: 20141217

REG Reference to a national code

Ref country code: LT

Ref legal event code: MG4D

PG25 Lapsed in a contracting state [announced via postgrant information from national office to epo]

Ref country code: LV

Free format text: LAPSE BECAUSE OF FAILURE TO SUBMIT A TRANSLATION OF THE DESCRIPTION OR TO PAY THE FEE WITHIN THE PRESCRIBED TIME-LIMIT

Effective date: 20141217

Ref country code: GR

Free format text: LAPSE BECAUSE OF FAILURE TO SUBMIT A TRANSLATION OF THE DESCRIPTION OR TO PAY THE FEE WITHIN THE PRESCRIBED TIME-LIMIT

Effective date: 20150318

REG Reference to a national code

Ref country code: NO

Ref legal event code: T2

Effective date: 20141217

PG25 Lapsed in a contracting state [announced via postgrant information from national office to epo]

Ref country code: RO

Free format text: LAPSE BECAUSE OF FAILURE TO SUBMIT A TRANSLATION OF THE DESCRIPTION OR TO PAY THE FEE WITHIN THE PRESCRIBED TIME-LIMIT

Effective date: 20141217

Ref country code: CZ

Free format text: LAPSE BECAUSE OF FAILURE TO SUBMIT A TRANSLATION OF THE DESCRIPTION OR TO PAY THE FEE WITHIN THE PRESCRIBED TIME-LIMIT

Effective date: 20141217

Ref country code: SK

Free format text: LAPSE BECAUSE OF FAILURE TO SUBMIT A TRANSLATION OF THE DESCRIPTION OR TO PAY THE FEE WITHIN THE PRESCRIBED TIME-LIMIT

Effective date: 20141217

Ref country code: EE

Free format text: LAPSE BECAUSE OF FAILURE TO SUBMIT A TRANSLATION OF THE DESCRIPTION OR TO PAY THE FEE WITHIN THE PRESCRIBED TIME-LIMIT

Effective date: 20141217

PG25 Lapsed in a contracting state [announced via postgrant information from national office to epo]

Ref country code: IS

Free format text: LAPSE BECAUSE OF FAILURE TO SUBMIT A TRANSLATION OF THE DESCRIPTION OR TO PAY THE FEE WITHIN THE PRESCRIBED TIME-LIMIT

Effective date: 20150417

Ref country code: PL

Free format text: LAPSE BECAUSE OF FAILURE TO SUBMIT A TRANSLATION OF THE DESCRIPTION OR TO PAY THE FEE WITHIN THE PRESCRIBED TIME-LIMIT

Effective date: 20141217

REG Reference to a national code

Ref country code: DE

Ref legal event code: R097

Ref document number: 602009028415

Country of ref document: DE

PLBE No opposition filed within time limit

Free format text: ORIGINAL CODE: 0009261

STAA Information on the status of an ep patent application or granted ep patent

Free format text: STATUS: NO OPPOSITION FILED WITHIN TIME LIMIT

26N No opposition filed

Effective date: 20150918

PG25 Lapsed in a contracting state [announced via postgrant information from national office to epo]

Ref country code: MC

Free format text: LAPSE BECAUSE OF FAILURE TO SUBMIT A TRANSLATION OF THE DESCRIPTION OR TO PAY THE FEE WITHIN THE PRESCRIBED TIME-LIMIT

Effective date: 20141217

REG Reference to a national code

Ref country code: FR

Ref legal event code: PLFP

Year of fee payment: 8

PG25 Lapsed in a contracting state [announced via postgrant information from national office to epo]

Ref country code: BE

Free format text: LAPSE BECAUSE OF FAILURE TO SUBMIT A TRANSLATION OF THE DESCRIPTION OR TO PAY THE FEE WITHIN THE PRESCRIBED TIME-LIMIT

Effective date: 20141217

PG25 Lapsed in a contracting state [announced via postgrant information from national office to epo]

Ref country code: MT

Free format text: LAPSE BECAUSE OF FAILURE TO SUBMIT A TRANSLATION OF THE DESCRIPTION OR TO PAY THE FEE WITHIN THE PRESCRIBED TIME-LIMIT

Effective date: 20141217

REG Reference to a national code

Ref country code: FR

Ref legal event code: PLFP

Year of fee payment: 9

PG25 Lapsed in a contracting state [announced via postgrant information from national office to epo]

Ref country code: HU

Free format text: LAPSE BECAUSE OF FAILURE TO SUBMIT A TRANSLATION OF THE DESCRIPTION OR TO PAY THE FEE WITHIN THE PRESCRIBED TIME-LIMIT; INVALID AB INITIO

Effective date: 20090630

PG25 Lapsed in a contracting state [announced via postgrant information from national office to epo]

Ref country code: CY

Free format text: LAPSE BECAUSE OF FAILURE TO SUBMIT A TRANSLATION OF THE DESCRIPTION OR TO PAY THE FEE WITHIN THE PRESCRIBED TIME-LIMIT

Effective date: 20141217

PG25 Lapsed in a contracting state [announced via postgrant information from national office to epo]

Ref country code: TR

Free format text: LAPSE BECAUSE OF FAILURE TO SUBMIT A TRANSLATION OF THE DESCRIPTION OR TO PAY THE FEE WITHIN THE PRESCRIBED TIME-LIMIT

Effective date: 20141217

REG Reference to a national code

Ref country code: FR

Ref legal event code: PLFP

Year of fee payment: 10

REG Reference to a national code

Ref country code: CH

Ref legal event code: PCAR

Free format text: NEW ADDRESS: SPLUEGENSTRASSE 10, 8002 ZUERICH (CH)

REG Reference to a national code

Ref country code: AT

Ref legal event code: UEP

Ref document number: 701978

Country of ref document: AT

Kind code of ref document: T

Effective date: 20141217

PG25 Lapsed in a contracting state [announced via postgrant information from national office to epo]

Ref country code: PT

Free format text: LAPSE BECAUSE OF FAILURE TO SUBMIT A TRANSLATION OF THE DESCRIPTION OR TO PAY THE FEE WITHIN THE PRESCRIBED TIME-LIMIT

Effective date: 20141217

REG Reference to a national code

Ref country code: HR

Ref legal event code: ODRP

Ref document number: P20150280

Country of ref document: HR

Payment date: 20190515

Year of fee payment: 11

REG Reference to a national code

Ref country code: HR

Ref legal event code: ODRP

Ref document number: P20150280

Country of ref document: HR

Payment date: 20200616

Year of fee payment: 12

REG Reference to a national code

Ref country code: HR

Ref legal event code: ODRP

Ref document number: P20150280

Country of ref document: HR

Payment date: 20210625

Year of fee payment: 13

REG Reference to a national code

Ref country code: HR

Ref legal event code: ODRP

Ref document number: P20150280

Country of ref document: HR

Payment date: 20220630

Year of fee payment: 14

PGFP Annual fee paid to national office [announced via postgrant information from national office to epo]

Ref country code: LU

Payment date: 20230525

Year of fee payment: 15

REG Reference to a national code

Ref country code: HR

Ref legal event code: ODRP

Ref document number: P20150280

Country of ref document: HR

Payment date: 20230526

Year of fee payment: 15

PGFP Annual fee paid to national office [announced via postgrant information from national office to epo]

Ref country code: NO

Payment date: 20230601

Year of fee payment: 15

Ref country code: NL

Payment date: 20230627

Year of fee payment: 15

Ref country code: IE

Payment date: 20230628

Year of fee payment: 15

Ref country code: FR

Payment date: 20230628

Year of fee payment: 15

Ref country code: DK

Payment date: 20230627

Year of fee payment: 15

Ref country code: DE

Payment date: 20230616

Year of fee payment: 15

Ref country code: BG

Payment date: 20230530

Year of fee payment: 15

PGFP Annual fee paid to national office [announced via postgrant information from national office to epo]

Ref country code: SI

Payment date: 20230531

Year of fee payment: 15

Ref country code: SE

Payment date: 20230628

Year of fee payment: 15

Ref country code: HR

Payment date: 20230526

Year of fee payment: 15

Ref country code: FI

Payment date: 20230627

Year of fee payment: 15

Ref country code: AT

Payment date: 20230627

Year of fee payment: 15

PGFP Annual fee paid to national office [announced via postgrant information from national office to epo]

Ref country code: IT

Payment date: 20230627

Year of fee payment: 15

Ref country code: GB

Payment date: 20230629

Year of fee payment: 15

Ref country code: ES

Payment date: 20230712

Year of fee payment: 15

Ref country code: CH

Payment date: 20230702

Year of fee payment: 15

PGFP Annual fee paid to national office [announced via postgrant information from national office to epo]

Ref country code: MK

Payment date: 20230531

Year of fee payment: 15

REG Reference to a national code

Ref country code: DE

Ref legal event code: R119

Ref document number: 602009028415

Country of ref document: DE

PG25 Lapsed in a contracting state [announced via postgrant information from national office to epo]

Ref country code: FI

Free format text: LAPSE BECAUSE OF NON-PAYMENT OF DUE FEES

Effective date: 20240630

REG Reference to a national code

Ref country code: HR

Ref legal event code: PBON

Ref document number: P20150280

Country of ref document: HR

Effective date: 20240630

PG25 Lapsed in a contracting state [announced via postgrant information from national office to epo]

Ref country code: BG

Free format text: LAPSE BECAUSE OF NON-PAYMENT OF DUE FEES

Effective date: 20240630

REG Reference to a national code

Ref country code: DK

Ref legal event code: EBP

Effective date: 20240630

PG25 Lapsed in a contracting state [announced via postgrant information from national office to epo]

Ref country code: FI

Free format text: LAPSE BECAUSE OF NON-PAYMENT OF DUE FEES

Effective date: 20240630

Ref country code: BG

Free format text: LAPSE BECAUSE OF NON-PAYMENT OF DUE FEES

Effective date: 20240630

REG Reference to a national code

Ref country code: CH

Ref legal event code: PL

REG Reference to a national code

Ref country code: NL

Ref legal event code: MM

Effective date: 20240701

REG Reference to a national code

Ref country code: AT

Ref legal event code: MM01

Ref document number: 701978

Country of ref document: AT

Kind code of ref document: T

Effective date: 20240630

PG25 Lapsed in a contracting state [announced via postgrant information from national office to epo]

Ref country code: LU

Free format text: LAPSE BECAUSE OF NON-PAYMENT OF DUE FEES

Effective date: 20240630

GBPC Gb: european patent ceased through non-payment of renewal fee

Effective date: 20240630

REG Reference to a national code

Ref country code: SE

Ref legal event code: EUG

PG25 Lapsed in a contracting state [announced via postgrant information from national office to epo]

Ref country code: NL

Free format text: LAPSE BECAUSE OF NON-PAYMENT OF DUE FEES

Effective date: 20240701

PG25 Lapsed in a contracting state [announced via postgrant information from national office to epo]

Ref country code: NL

Free format text: LAPSE BECAUSE OF NON-PAYMENT OF DUE FEES

Effective date: 20240701

PG25 Lapsed in a contracting state [announced via postgrant information from national office to epo]

Ref country code: DE

Free format text: LAPSE BECAUSE OF NON-PAYMENT OF DUE FEES

Effective date: 20250101

Ref country code: HR

Free format text: LAPSE BECAUSE OF NON-PAYMENT OF DUE FEES

Effective date: 20240630

PG25 Lapsed in a contracting state [announced via postgrant information from national office to epo]

Ref country code: IE

Free format text: LAPSE BECAUSE OF NON-PAYMENT OF DUE FEES

Effective date: 20240630

PG25 Lapsed in a contracting state [announced via postgrant information from national office to epo]

Ref country code: NO

Free format text: LAPSE BECAUSE OF NON-PAYMENT OF DUE FEES

Effective date: 20240630

PG25 Lapsed in a contracting state [announced via postgrant information from national office to epo]

Ref country code: AT

Free format text: LAPSE BECAUSE OF NON-PAYMENT OF DUE FEES

Effective date: 20240630

Ref country code: CH

Free format text: LAPSE BECAUSE OF NON-PAYMENT OF DUE FEES

Effective date: 20240630

PG25 Lapsed in a contracting state [announced via postgrant information from national office to epo]

Ref country code: FR

Free format text: LAPSE BECAUSE OF NON-PAYMENT OF DUE FEES

Effective date: 20240630

PG25 Lapsed in a contracting state [announced via postgrant information from national office to epo]

Ref country code: IT

Free format text: LAPSE BECAUSE OF NON-PAYMENT OF DUE FEES

Effective date: 20240630

Ref country code: GB

Free format text: LAPSE BECAUSE OF NON-PAYMENT OF DUE FEES

Effective date: 20240630

PG25 Lapsed in a contracting state [announced via postgrant information from national office to epo]

Ref country code: DK

Free format text: LAPSE BECAUSE OF NON-PAYMENT OF DUE FEES

Effective date: 20240630

REG Reference to a national code

Ref country code: ES

Ref legal event code: FD2A

Effective date: 20250805

REG Reference to a national code

Ref country code: SI

Ref legal event code: KO00

Effective date: 20240701

PG25 Lapsed in a contracting state [announced via postgrant information from national office to epo]

Ref country code: ES

Free format text: LAPSE BECAUSE OF NON-PAYMENT OF DUE FEES

Effective date: 20240701

PG25 Lapsed in a contracting state [announced via postgrant information from national office to epo]

Ref country code: SE

Free format text: LAPSE BECAUSE OF NON-PAYMENT OF DUE FEES

Effective date: 20240701